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Wei-Kang Wu, Lindsay B. Nicholson, Dave O. Bates, Andrew D. Dick; IL-4 Stimulates Macrophage Soluble Flt-1 Production and IL-4 Administration Inhibits Laser-induced CNV in Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2302.
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Activation and infiltration of myeloid derived cells participates in retinal angiogenesis and is central to tissue remodeling. Their exact role during pathological angiogenesis remains undefined. Murine macrophages activated by Th2 cytokines (IL-4 and IL-13) display anti-angiogenic properties, notably increasing soluble VEGF receptor-1 (sFlt-1)/ VEGF ratio in vitro. To understand whether there is an inhibitory role for IL-4 in vivo we studied its administration in laser-induced choroidal neovascularization (LI-CNV).
Bone marrow derived macrophages (BMDMs) were conditioned with either IL-4 (20U/ml) or IL-13 (20U/ml, M2 signature) or PGE2 (50ng/ml, pro-angiogenic; VEGFhi M2 signature), and sFlt-1 mRNA and protein expression were determined. Macrophage sFlt-1-mediated VEGF neutralization was confirmed by a modified binding assay. HUVEC proliferation was estimated by alamarBlue staining to assess the bioactivity of sFlt-1 induced by IL-4 or IL-13, and specificity of sFlt-1 inhibition was confirmed following sFlt-1 siRNA knockdown as well in cytokine-depleted culture conditions. The model of LI-CNV in C57BL/6 mice was used to examine whether intravitreal administration of 0.6µg IL-4 per eye inhibits angiogenesis.
IL-4 or IL-13-induced macrophage sFlt-1 expression (23.4±1.3 fold). Specific sFlt-1 binding to VEGF and inhibition of HUVEC proliferation was observed in IL-4 or IL-13-conditioned media and specificity confirmed through the absence of response following sFlt-1 knockdown. IL-4, IL-13 and PGE2 all generate M2 macrophages (arginase-1hi). However, PGE2 conditioning increased VEGF (2.72±0.32 fold) which was reduced by IL-4 (to 12.4±4.6%) or IL-13 (to 52.5±5.9%), and IL-4 or IL-13-induced sFlt-1 production was inhibited by PGE2 (to 12.0±1.8%). Angiogenesis was decreased in IL-4 treated eyes following LI-CNV. To confirm IL-4 effect, administration of 20µg per eye anti-IL-4 mAb increased CNV area.
We have shown that IL-4 and IL-13 induce macrophage sFlt-1 and inhibit PGE2-mediated pro-angiogenic property of macrophages. IL-4 administration inhibits angiogenesis, and IL-4 neutralization during CNV increases neovascularization, suggesting that endogenous IL-4 plays a role limiting vessel growth.
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